It is known that immunoglobulin (Ig) is found in the body fluids of all vertebrate animals from fish to mammals, and is produced by lymphoid cells. It is classified into five classes, namely, IgG, IgM, IgA, IgD and IgE, according to the physicochemical and immunological properties, wherein the fundamental structure of molecules are common to each class, and is composed from H chain having fifty to seventy thousand molecular weight and L chain having twenty-three thousand molecular weight, and the H chain is structured with γ, μ, α, δ, and ε chain corresponding to IgG, IgM, IgA, IgD and IgE. The two H chains from the hinge region to the C terminal obtained by decomposing this Ig molecule with papain, which are bound by S—S binding is called a Fc fragment, and the receptor on the cell surface which this Fc fragment binds to is called a Fc receptor (hereinafter “FcR”). It is known that these FcRs derive signaling in the cells and crosslink by antigen-antibody complex which is a ligand, thereby constructing a hematopoietic cell surface molecule family that induces the response of many effectors. Further, it is known that FcR, which shows affinity to the Fc region of Ig and exists on the cell surface, is involved in immunoresponses such as antibody-dependent cytotoxic reaction and hypersensitive reaction.
The types of FcRs known are Fcγ receptor that binds specifically to the γ chain of IgG in the body fluid, Fcε receptor that binds specifically to the ε chain of IgE, FcαR that binds specifically to the α chain of IgA, and the like. It is known that the Fc receptors of these immunocompetent cells have a significant relationship with cell function, and that many of them exist in the large portion of lymphocyte, a part of the T cell lymphocyte, mononuclear cell, neutrophil, basophil, macrophage, mastocyte (mast cell), platelet or the like. Further, these receptors play a role in the function of lymphocyte that is antibody-dependent, however, its inherent function is poorly understood. Fcγ receptor (hereinafter “FcγR”) is classified mainly into three types, type I (CD64 antigen), type II (CD 32 antigen) and type III (CD 16 antigen), based on the similarity of gene structure. It is known that FcγRI is a glycoprotein with a molecular weight of 72 kDa, which binds to the IgG monomer with high affinity, and expresses on the monocyte and macrophage. FcγRII differs from the other FcRs in that it has low affinity to the IgG monomer, binds to the polyvalent IgG that has become an immunocomplex, and is widely expressed in the hematopoietic stem cells including monocyte, macrophage, polymorphonuclear (PMN) leukocyte, mast cell, platelet, some of the T cell lymphocytes and B cell lymphocytes. Further, it is known that FcγRIII is a low-affinity FcγR, which expresses in natural killer cell, macrophage, PMN and mast cell. It is also known that three types of receptors having different gene arrangements, FcγRIIA, FcγRIIB and FcγRIIC, exist in FcγRII, and that each of its chromosome is positioned in 1q23.
Recently, knockout mice for these FcR molecular groups have been generated continuously (Cell 75, 969-976, 1993; Cell 76, 519-529, 1994; Nature 379, 346-349, 1996; Immunity 5, 181-188, 1996; Nature 369, 753-756, 1994; J. Immunol. 152, 3378-3390, 1994; Proc. Natl. Acad. Sci. USA 91, 6835-6839, 1994), and the understanding of the physiological function of some of the FcRs have progressed. Moreover, the present inventors have generated a knockout mouse of FcR γ chain isolated and identified as a homodimeric molecule that is associated with FcεRI, which is a high-affinity receptor of IgE (Cell, 76, 519-529, 1994). It is also known that this FcRγ chain knockout mouse is deficient in the expression and function of FcR, at least in the three types of FcγRI, FcγRIII and FcεRI.
The FcR that expresses in the immunocytic group mentioned above is a molecule that begins various immunoreaction and inflammatory response to antibody dependency. Since FcR expresses by duplicating in multiple cell species, the role of individual FcRs became clear only after several mice whose FcR expression is genetically deficient was generated. Presently, it has been revealed that the role of FcR is involved in the onset of diseases such as allergy (Cell 75, 969-976, 1993; Cell 76, 519-529, 1994), Arthus reaction (Science 265, 1095-1098, 1994; Immunity 5, 387-390), rheumatoid arthritis (J. Exp. Med. 189, 187-194, 1999; J. Exp. Med. 191, 1611-1616, 2000), immunologic glomerulonephritis (Kidney Int. 54, 1166-1174, 1998; Eur. J. Immunol. 30, 1182-1190, 2000), autoimmune vasculitis (Blood 94, 3855-3863, 1999), systemic lupus erythematosus (Science 279, 1052-1054, 1998) and Goodpasture's syndrome (Goodpasture's; J. Exp. Med. 191, 899-906, 2000), as well as biophylaxis. Moreover, the essential role of FcR in the various immune processes is progressively elucidated by the finding of the mutate mouse mentioned above (Annu. Rev. Immunol. 16, 421-432, 1998; Annu. Rev. Immunol. 15, 203-234, 1997; Takai, T., and J. V. Ravetch. 1998. Fc receptor genetics and the manipulation of genes in the study of FcR biology. In Immunoglobulin Receptors and their Physiological and Pathological Roles in Immunity. J. G. J. van de Winkel and P. Mark Hogarth, editors. Kluwer Academic Publishers, Netherland, 37-48).
It has also been reported that the FcγRIII on the mast cell plays a primary role at the start of the two types of distinct immunoresponses, that is, IgG-dependent anaphylaxis (J. Clin. Invest. 99, 915-925, 1997; J. Clin. Invest. 99, 901-914, 1997; J. Exp. Med. 189, 1573-1579, 1999) and passive Arthus reaction (Science 265, 1095-1098, 1994; Immunity 5, 387-390, J. Exp. Med. 184, 2385-2392, 1996; Immunity 5, 181-188, 1996; Eur. J. Immunol. 30, 481-490, 2000). These immunoresponsive models are distinguished from one another by the difference of the period of development of the disease and histology. In anaphylaxis, skin edema and systemic blood supply failure (shock) are developed immediately after antigen exposure. In contrast, Arthus reaction is found a few hours after antigen exposure, as a tissue damage accompanied by hemorrhage. Presently, the formation of pathology of models that indicate these characteristics, are explained as a result of time-limited discharge of inflammatory mediator, for example, the preexisting substances such as histamine and serotonin, and substances that are newly synthesized such as arachidonic acid metabolite and cytokine. However, the intracellular mechanism involved in the development of pathology of these models remains unclear.
Meanwhile, it is known that Lyn belongs to the Src family kinase, associates with a variety of immunoreceptors that have ITAM motif (an amino acid sequence that is recognized and bound by the SH2 region of the intracellular tyrosine kinase), and has a function to begin intracellular signaling (Int. J. Biochem. Cell. Biol. 29, 397-400, 1997; Annu. Rev. Immunol. 17, 555-592, 1999). In a recent research using a Lyn deficient mouse, it was revealed that Lyn plays an essential role in the individual body, for B cells and mast cells to function normally (Cell 83, 301-311, 1995; Immunity 3, 549-560, 1995; Immunity 7, 69-81, 1997). In fact, in a Lyn deficient mouse, marked damage is shown in the cutaneous anaphylaxis reaction which is induced by IgE and antigen, and Lyn plays an important role in the signaling through FcεRI (J. Immunol. 158, 2350-2355, 1997).
As mentioned above, Lyn tyrosine kinase, which belongs to the Src family, associates functionally with a variety of receptors such as FcR and the like, and plays a significant role in intracellular signaling. In contrast, in an inflammatory response such as autoimmune disease, allergy reaction or the like, wherein the immunocomplex is involved, the role of the FcγR of IgG expressed in mast cells and the like are drawing attention. For example, it was revealed by research using knockout mice that FcγRIII activates mast cells through the immunocomplex, and on the contrary, FcγRIIB inhibits mast cells. However, it was not possible to make an evaluation of type III allergy inflammation independently, since normally, the FcγRIIB shows inhibitory action. An object of the present invention is to provide an experimental model animal that does not develop anaphylaxis, a type I allergy, can specifically induce Arthus reaction, a type III allergy, and can independently evaluate the type III allergy inflammation without being affected by type I allergy, and a method of screening a reaction accelerating or inhibitory substance in a type III allergy reaction through FcγRIII using said experimental model animal.
The inventors of the present invention have conducted intensive study to attain the object mentioned above, and generated a double deficient mouse of Lyn and FcγRIIB in order to eliminate FcγRIIB which inhibits the response through FcγRIII. The significance of the role of Lyn in the downstream of FcγRIII signaling was studied by inducing systemic passive anaphylaxis with IgG-immunocomplex. From the fact that systemic passive anaphylaxis is Lyn-requisite and reverse passive Arthus reaction is not Lyn-requisite, the pathway connected to the allergy response through FcγRIII has two pathways, Lyn-dependent and Lyn-independent, and the inventors focused attention on the function of Lyn-independent pathway in type III allergy reaction. Thus, the present invention had been completed.